This invention relates to peptide-based compounds having light-emitting moieties. Peptides may be chemically linked with detectable xe2x80x9clabelsxe2x80x9d and used as probes, for example, to monitor peptide, cytokine, drug, and hormone receptors at the cellular level. Typically, the labeled peptide is placed in contact with a tissue or cell culture where it binds to an available receptor. Once bound, the label is detected, allowing properties such as receptor distribution or receptor binding kinetics to be monitored.
Peptides are typically labeled with radioactive elements such as 125I or 3H. In this case, emission of high-energy radioactive particles is monitored using standard xcex3-ray detectors, thereby allowing detection of the label. While detection techniques for 125I and 3H are well-known, radioactive compounds by nature have limited half lives, and are often both toxic and expensive. Moreover, current detection technology makes it difficult or impossible to detect radioactive probes in real-time, thereby precluding study of kinetic processes.
Peptides labeled with non-radioactive molecules have also been disclosed. Macielag et al. (Int. J. Pept. Protein Res. (1994) 44(6):582-588)) discloses a biotin-labeled motilin peptide. However, detection of this biotin-labeled motilin peptide requires and multiple assay steps.
Motilin is a 22-amino acid polypeptide secreted from the endocrine cells of the mucosa of the upper part of the small intestine. Motilin binds to G-coupled receptors to stimulate peristaltic contractions of the stomach and small intestine during fasting (Lee et al., Am. J. Physiol. (1983) 245G547-G553; Poitras , P. Gastroenterology (1984) 87:909-913; Scarpignato et al, Can. J. Gastroenter. (1999) 13(A): 50A-65A; Usellin et al., Histochemistry (1984) 81:363-368). There is also evidence that suggests motilin and motilin receptors are present in the brain and have a role in the regulation of food intake (Asakawa et al., Peptides (1998) 19(6):987-990).
Motilin""s wide tissue distribution makes it a particularly desirable peptide to label and use to monitor cell receptors, as these peptides exhibit multiple biological roles and their receptors are located in a variety of tissues. Labeled motilin polypeptides are valuable in the identification of agonists and antagonist of motilin which may be useful as appetite stimulants or in the treatment of digestive disorders, for example, hypokinetic and hyperkinetic digestive disorders, such as irritable bowel syndrome associated constipation or diarrhea, or Chron""s disease.
There exists the need for peptides that are chemically linked to detectable labels that are easily detected, yet do not decrease the biological activity of the peptide.
The present invention provides a compound containing a motilin peptide and a light-emitting moiety that is both biologically active and optically detectable. The peptide is chemically attached to the light-emitting moiety at an amino acid position that is not involved in binding to the peptide receptor. In this way, the peptide""s affinity for the binding site is not significantly decreased and the compound retains high biological activity. Furthermore, the compound can be easily detected using standard optical means.
In general, in one aspect, the invention provides a biologically active compound of the formula: 
where R1 is a light-emitting moiety and R2 is a motilin peptide, fragment, derivative or analog thereof The peptide is linked at a first amino acid position to (Cxe2x80x94X) which, in turn, is selected from the group including Cxe2x95x90O, Cxe2x95x90S, CH(OH), Cxe2x95x90Cxe2x95x90O, Cxe2x95x90NH, CH2, CH(OR), CH(NR), CH(R), CR3R4, and C(OR3)OR4 where R, R3, and R4 are alkyl moieties or substituted alkyl moieties. Optionally the compound may include a linker moiety between the peptide and the Cxe2x80x94X binding group. Preferably, the compound exhibits substantial biological activity in the presence of receptors having affinities for motilin peptides. The compound may also be in the form of a pharmaceutically acceptable salt or complex thereof. Preferably, one of the C-terminal eight residues of the motilin peptide of SEQ ID NO:1(referred to herein is the C-terminal octapeptide) that is not required for motilin receptor binding or stability is attached to (Cxe2x80x94X), either directly or through a linker moiety (Macielag et al., supra).
In preferred embodiments, the motilin peptide can be any peptide that shares sufficient homology with SEQ ID NO:1, Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln or a fragment, derivative, or analog thereof. In particularly preferred embodiments, motilin peptides homologs, such as the canine motilin SEQ ID NO:2, Phe-Val-Pro-Ile-Phe-Thr-His-Ser-Glu-Leu-Gln-Lys-Ile-Arg-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gly (MW 2685) and porcine motilin SEQ ID NO:3, Phe-Val-Pro-Ile-Phe-Thr-Tyr-Gly-Glu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln (MW 2697) may be used as motilin peptides in the present invention. Alternatively, the motilin peptide could be a modified motilin peptide that includes various substitutions or contains modified amino acids, such as beta-alanine or norleucine and the like. In preferred embodiments, any one of the eight residues of the C-terminal octapeptide of motilin is preferably chemically bound to the (Cxe2x80x94X) moiety of the present invention. In one preferred embodiment, the motilin peptide is chemically bound to the (Cxe2x80x94X) moiety through Lys20. In a particularly preferred embodiment, the peptide is attached to the (Cxe2x80x94X) moiety through Lys20 and the (Cxe2x80x94X) bond is preferably either Cxe2x95x90O or Cxe2x95x90S. In yet another preferred embodiment, the peptide may be amidated at the C-terminus.
In other preferred embodiments, the light-emitting moiety (R1) is selected from the group including 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (e.g., 6-((4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora-3a,4a-diaza-s-indacene-2-propionyl)amino) hexanoic acid) (BODIPY(copyright) TMR) or 4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY(copyright) 576/589)), fluorescein, fluorescein isothiocynate (FITC, Texas red, phycoerythrin, rhodamine, carboxytetramethylrhodamine, indopyras dyes, Cascade blue, coumarins, nitrobenzo-2-oxa-diazole (NBD), Lucifer Yellow, propidium iodide, CY3, CY5, CY9, dinitrophenol (DNP), lanthanide cryptates, lanthanide chelates, non-fluorescent dialdehydes (OPA, NDA, ADA, ATTOTAG reagents from Molecular Probes) which react with primary amines (N-term lys) in the presence of a nucleophile (i.e. CN) to form fluorescent isoindoles, dansyl dyes fluorescamine and dabcyl chloride, 5-((((2-iodoacetyl)amino)ethyl)amino) naphthalene-1-sulfonic acid, long lifetime dyes comprised of metal-ligand complexes (MLC) which consist of a metal center (Ru, Re, Os) and organic or inorganic ligands complexed to the metal such as [Ru(bpy)3]2+ and [Ru(bpy)2(dcbpy)], and the like and derivatives thereof. The light-emitting moiety can be attached to the peptide by reaction of a reactive side group (of a light-emitting molecule) with any C-terminal eight residues of the motilin peptide. Suitable reactive side groups include, by way of example only, indoacetamide, maleimide, isothiocyanate, succinimidyl ester, sulfonyl halide, aldehydes, glyoxal, hydrazine and derivatives thereof.
The above-identified compound is useful in the labeling of cell receptor sites, cell sorting, flow cytometry and performing fluoroimmunoassays. In another aspect, the invention provides a method for labeling a receptor having an affinity for a motilin peptide by contacting the receptor with one or more of the compounds described above. Cell receptor sites, can be imaged by contacting candidate cell receptor sites with the compound of the invention, and then detecting the bound compounds as an indication of the cell receptor sites. Cell sorting can be performed by contacting a population of cells with compound and isolating cells bound to the compound. Flow cytometry can be performed by contacting a population of cells with the compound and detecting cells bearing receptors on their surfaces by detecting cells bound to the compound. In other preferred embodiments it is possible to use the compound to label receptor sites on a model organism (e.g., C. elegans) in order to elucidate the tissue distribution of the receptor sites.
The invention has many advantages. In a general sense, peptide-containing compounds which retain their biological activity after being labeled with light-emitting moieties have a wide variety of biological applications. Such compounds can be used to identify, visualize, quantify, target, and select receptors on cells and tissues both in vitro and in vivo. These compounds may be used in place of more conventionally labeled peptides, such a 125I radiolabeled peptides. Radiolabeled compounds are often toxic, environmentally hazardous, chemically unstable and have, by the nature of the radioactive decay rate, relatively short lifetimes. In contrast, fluorescently-labeled motilin peptides are relatively safe and non-toxic, thereby allowing it to be synthesized and used without employing special laboratory procedures. Similarly, following use, fluorescent motilin peptides may be easily disposed, whereas disposal of radioactive compounds is both time-consuming and costly. In addition, fluorescent markers for motilin peptide receptors are stable and may be stored for extensive periods of time without undergoing considerable degradation.
Use of motilin peptides in the labeled compound provides several additional advantages. As described above, motilin peptides exhibit biological activity in tissues such as the brain and the gastrointestinal tract, and are involved in the regulation of gastrointestinal motor activity during fasting. Therefore, motilin peptides may be used as probes to investigate a number of different cell types. In addition, the motilin peptide has a relatively simple structure (10-27 amino acids) and can be synthesized and isolated with standard, well-known techniques.
During typical experiments, fluorescent markers for motilin receptors emit optical signals, and thus may be monitored by eye or with the aid of external optical detectors. In this way, the fluorescent peptides obviate the need for secondary detection steps sometimes used for radiolabeled compounds or incubation with secondary labeled compounds. Detection of optical radiation is, in general, relatively simple and cost-effective compared to detection of radioactive particles (e.g., xcex1-particles); conventional charge-coupled device (CCDs) or light-sensitive cameras can therefore be used without modification for this application.
In addition, because of their high optical emission rates and well-characterized emission cross sections, fluorescent markers attached to motilin peptides can be used for real-time, quantified imaging of a number of dynamic biological phenomena, such as kinetics associated with receptor binding. The compounds can also be used for static processes, such as monitoring peptide distribution within a cell. Motilin peptide receptors marked with fluorescent markers may also be used in flow cytometry, confocal microscopy, fluorescence polarization spectroscopy, and any other techniques exploiting the optical detection of fluorescence or photoluminescence.
In a related aspect the present invention provides an assay method for evaluating a known or candidate motilin peptide for receptor binding that involves bringing together a molecule, the labeled motilin compound of the invention, and a motilin receptor preparation containing motilin receptors capable of detectably binding motilin, and determining or measuring the ability of the molecule to compete against the compound for binding to the motilin receptor preparation. A similar assay method is provided by the invention for determining the presence or amount of a motilin receptor binding molecule in a test sample that includes the steps of a) bringing together the test sample and a motilin receptor preparation containing motilin receptors capable of detectably binding motilin; b) measuring the ability of the test sample to compete against the labeled motilin compound the invention for binding to the motilin receptor preparation; and c) optionally comparing the amount of motilin receptor binding molecule in the test sample to the amount of motilin receptor binding in a control sample.
In another related aspect, the invention provides an assay method for screening cell lines, cells desegregated from tissue, or cell membrane preparations, to identify those cells that carry motilin, that involves a) contacting test cells or cell membrane preparations with the labeled motilin compound of the invention, and b) detecting an increase in fluorescent signal on the cell compared to a control, an increase in fluorescent signal indicating that a motilin receptor is present in the cell.
In a final aspect, the invention provides a kit for identifying natural and non-natural molecules that bind to the human motilin receptor, for use in treating motilin-related disease that includes a) at least one of the labeled motilin compounds of the invention; and b) a receptacle containing a human motilin receptor preparation. Optionially, the kit further includes instructions for utilizing the reagents in (a) and (b) in fluorescence assays to identify the molecules.
Other advantages and features of the invention will become apparent from the following detailed description, and from the claims.
xe2x80x9cMotilin peptidexe2x80x9d: xe2x80x9cMotilin peptidexe2x80x9d In preferred embodiments, the motilin peptide can be any peptide that shares sufficient homology with motilin (SEQ ID NO:1). In particularly preferred embodiments, the motilin peptide is the canine (SEQ ID NO:2) or porcine (SEQ ID NO:3) motilin peptide. For example, any peptide with substantial homology and biological activity similar to motilin, as determined by one of ordinary skill in the art, would be considered to qualify as a motilin peptide. For example, according to the present invention, a motilin peptide has preferably 60% amino acid identity to motilin, more preferably, 80% amino acid identity, most preferably, 90% amino acid identity over a span of at least 10 amino acids. Preferably, the motilin peptide or xe2x80x9cmotilin compoundxe2x80x9d includes fragments of motilin, derivatives, or analogs thereof. Motilin peptides may be peptides whose sequences differ from motilin""s wild-type sequence by only conservative amino acid substitutions. For example, one amino acid may be substituted for another with similar characteristics (e.g., valine for glycine, arginine for lysine, etc.) or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not abolish the peptide""s biological activity. Alternatively, the motilin peptide could be a modified motilin peptide that includes various substitutions or contains modified amino acids, such as beta-alanine or norleucine and the like. Other useful modifications include those which increase motilin""s stability. The peptide may contain, for example, one or more non-peptide bonds (which replace a corresponding peptide bond) or D-amino acids in the peptide sequence. Additionally, the C-terminus carboxylic acid group may be modified to increase peptide stability. For example, as described above, the C-terminus may be amidated or otherwise derivatized to reduce the peptide susceptibility to degradation.
xe2x80x9cReceptorxe2x80x9d: As referred to herein, the term xe2x80x9creceptorxe2x80x9d or xe2x80x9cmotilin receptorxe2x80x9d refers to a macromolecule capable of recognizing and specifically binding with a motilin ligand, and which after binding, is capable of stimulating or suppressing the physical or chemical signal that initiates the chain of events leading to a physiological response from that receptor (Blecher et al. xe2x80x9cReceptors and Human Disease,xe2x80x9d Williams and Wilkins, Baltimore, 1981, Chapter 1). It is thus an important part of this invention that the tissues, cells, membranes, and recombinant receptor preparations used in the present invention exhibit the binding characteristics of a natural receptor.
The term xe2x80x9cnaturalxe2x80x9d may also be use to refer to a molecule to differentiate a natural molecule from a synthetic molecule (i.e., a recombinant molecule). Essentially, xe2x80x9cnaturalxe2x80x9d means occurring in nature.
xe2x80x9cFluorescent peptidexe2x80x9d or xe2x80x9ccompoundxe2x80x9d: As referred to herein, xe2x80x9cfluorescent peptidexe2x80x9d or xe2x80x9ccompoundxe2x80x9d refers to a peptide-based compound that has been labeled with a light emitting moiety. The fluorescent peptide has the desirable characteristics of preserving the biological activity of the peptide-based compound, for example in receptor binding, and providing a detectable signal that can be measured using standard optical means.
xe2x80x9cLight emitting moleculexe2x80x9d: xe2x80x9cLight emitting molecule,xe2x80x9d as used herein, refers to a molecule capable of emitting light of any detectable wavelength that is not attached to a peptide of the present invention and may include a reactive side chain for coupling with a motilin peptide.
xe2x80x9cLight emitting moietyxe2x80x9d: xe2x80x9cLight emitting moietyxe2x80x9d is used to refer to a light emitting molecule (e.g., a fluorescent dye) that has been attached by any of a variety of means, as described below, to peptide-based moiety. Attachment to the peptide-based moiety is carried out so that the biological activity of the peptide-based moiety is maintained. The light emitting moiety provides a detectable signal of a particular wavelength. In general, the signal provided by light emitting moieties may be detected by a variety of techniques including conventional microscopy methods, including fluorescence or confocal microscopy, atomic force microscopy, fluorescence polarization spectroscopy and fluorimetry. Particularly preferred light emitting moieties are described in more detail below.
xe2x80x9cPeptide moietyxe2x80x9d: xe2x80x9cPeptide moietyxe2x80x9d, as used herein, refers to any peptide composed of any sequence of natural and/or custom amino acids. By xe2x80x9ccustom amino acidxe2x80x9d is meant any amino acid that cannot be found in nature, but can be synthesized in a laboratory. Such amino acids are often chemically modified amino acids. It is well known that natural amino acids may also be synthesized. Particularly preferred peptide moieties of the present invention include motilin peptides. Most particularly preferred are the canine and porcine motilin peptides or fragments, derivatives, or analogs thereof.
xe2x80x9cLinker moietyxe2x80x9d or xe2x80x9clinkerxe2x80x9d: A xe2x80x9clinker moietyxe2x80x9d or xe2x80x9clinkerxe2x80x9d is any moiety of the compound located between the peptide and the label or at any other position which provides greater three dimensional separation between the label and the peptide. Moieties that may be used as linkers in the present invention include those derived from glycine, xcex3-aminobutyric acid, beta-alanine, aminopentanoic acid, aminohexanoic acid, aminohepanoic acid, aminooctanoic acid, aminononanoic acid, aminodecanoic acid, aminoundecanoic acid, and aminododecanoic acid. Each of these moieties include an amino and a carboxylic acid functionality and so may be incorporated into the compound using a peptide bond.
xe2x80x9cBiologically active compoundxe2x80x9d or xe2x80x9cbiologically active peptidexe2x80x9d: xe2x80x9cBiologically active compoundxe2x80x9d or xe2x80x9cbiologically active peptidexe2x80x9d, as used herein, refers to the fluorescently labeled peptide of the invention represented in the formula described below and in FIG. 1. Any biologically active compound of the present invention is substantially biologically active.
xe2x80x9cSubstantially biologically activexe2x80x9d: In all cases, by xe2x80x9csubstantially biologically activexe2x80x9d is meant that the compound binds to a receptor having an affinity IC50 or Ki value for the compound which is no more than 100 times, preferably no more than 15 times, more preferably no more than 10 times and most preferably equal to or less than that of the corresponding unlabeled peptide. Most preferably, an affinity IC50 or Ki value for the compound is no more than 10 nM. Receptor affinity in this case can be determined using known methods, such as methods involving competitive binding of radioactively labeled peptides or by using known methods of fluorescence polarization or other known fluorescence technique for measuring the Kd for the receptor/peptide interaction.
xe2x80x9cLowxe2x80x9d or xe2x80x9cnoxe2x80x9d biological activity or xe2x80x9cbiologically inactivexe2x80x9d: By xe2x80x9clowxe2x80x9d or xe2x80x9cnoxe2x80x9d biological activity or xe2x80x9cbiologically inactivexe2x80x9d is meant biological activities less than 0.25% of the biological activity of R2xe2x80x94H in the presence of a receptor having affinity for motilin peptides.
xe2x80x9cMotilin-related diseasexe2x80x9d: By xe2x80x9cmotilin-related diseasexe2x80x9d is meant a disease that is at least in part caused by a defect in motilin activity (e.g., motilin receptor binding).